Protein-based therapeutic drugs have demonstrated significant efficacy in controlling and curing disease. Unlike traditional small molecule-based drug therapies, a major hurdle in the development of protein drugs is the challenge of maintaining the protein in the folded state throughout processing and also during storage at the end point-of-use. Patient compliance decreases as the complexity of administration increases, consequently liquid formulation of parenterals is highly preferred over lyophilized dosages that require reconstitution by the patient. Ionic liquids (ILs) are a class of materials that have considerable potential to provide advances in liquid formulation of protein pharmaceuticals. ILs is materials that have ionic character yet are in liquid form at room temperature. Although traditional ILs are synthesized from imidazolium-based actions and highly fluorinated anions, ILs can also be formulated from salts, sugars, amino acids, and biomolecules that exist in nature - many of which have already been approved as excipients. The exciting feature of ILs is that, because they consist of chemically distinct ions, the hydrogen-bonding character and the water miscibility can effectively be `tuned' to the application depending on the nature of the action and anion in the mixture. The following specific aims represent the critical steps in identifying potential ILs for use as protein stabilizers in liquid formulations: Aim #1: To evaluate the cyto-toxicity and water miscibility of a panel of rationally designed ILs to identify the least toxic, most hydrophilic compounds for continued studies. Aim #2: To identify the most solubilizing ILs for cytochrome C, lysozyme, and bovine serum albumin from the subset of ILs prioritized in Aim #1. Aim #3: To determine if the selected ILs can preserve protein structure and function in liquid formulation over long periods of storage and also under short term physical and chemical stresses. Broader Impact: The proposed work fits well with the goals of the NIH because it will advance our understanding of protein stabilization in liquids as it pertains to drug formulation. Furthermore, successful achievement of the proposed goals will support, promote, and sustain advances in the economical formulation of protein-based therapeutics for the treatment of human disease. This will have a direct and immediate impact on national health and welfare issues. PUBLIC HEALTH RELEVANCE: Protein-based therapeutic drugs have demonstrated significant efficacy in controlling and curing disease. Unlike traditional small molecule-based drug therapies, a major hurdle in the development of protein drugs is the challenge of maintaining the protein in the folded state throughout processing and also during storage at the end point-of-use. Ionic liquids (ILs) are a class of materials that have considerable potential to provide advances in liquid formulation of protein pharmaceuticals. This project involves the synthesis, characterization, and optimization of newly designed biocompatible ionic liquids to facilitate solubilization and stabilization of proteins, and ultimately stable long-term storage and delivery of protein-based therapeutics. The proposed work will advance our understanding of protein stabilization in liquids as it pertains to drug formulation. Furthermore, successful achievement of the proposed goals will support, promote, and sustain advances in the economical formulation of protein-based therapeutics for the treatment of human disease. This will have a direct and immediate impact on national health and welfare issues. [unreadable] [unreadable] [unreadable]